Team:Goettingen/week16-3

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<b>V08_16_1 2<sup>nd</sup> round: Purification of ligation from V08_15</b><br>
<b>V08_16_1 2<sup>nd</sup> round: Purification of ligation from V08_15</b><br>
<ul>
<ul>
-
<li>Experiment: <br>The purification of the ligation was now done using the peqGOLD Cycle-pure Kit (PeqLab) instead of an ethanol precipitation. </li>
+
<li>Experiment: <br>The purification of the ligation was now done using the peqGOLD Cycle-pure Kit (PeqLab) with columns from peqGOLD Plasmid Miniprep Kit I (PeqLab) instead of an ethanol precipitation. This way we could be sure to gain enough DNA material to go on with our library generation. Elution in 50 µL EB.</li>
</ul>
</ul>
<ul>
<ul>
-
<li>Observations & Results: <br>We now seemed to have found the mistake that caused problems with the loss of DNA material in the second mutagenesis round! Miscommunication led to using the wrong kit for purification steps. We decided to use the peqGOLD Cycle-pure Kit (PeqLab) instead of the subsequent ethanol precipitation as well, as this seemed to rise fewer problems.</li>
+
<li>Observations & Results: The corresponding agarose gel FINALLY showed a band of the expected size, indicating that we could purify our DNA material successfully.<br></li>
</ul>
</ul>
<br>
<br>
-
<b>V08_16_2 2<sup>nd</sup> round: Ligation</b><br>
+
<b>V08_16_2 2<sup>nd</sup> round: Transformation of electrocompetent cells with mutant plasmid mixture</b><br>
<ul>
<ul>
-
<li>Experiment: <br>The PCR purification was performed using the peqGOLD Cycle-pure Kit (PeqLab), modified using columns from the peqGOLD Plasmid Miniprep Kit (PeqLab)! Elution in 100 µL EB.</li>
+
<li>Experiment: <br>Electrocompetent cells were prepared according to <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>. Transformation was performed acording to the established protocol from <a href="https://2012.igem.org/Team:Goettingen/week10-3">week 10</a>.</li>
-
</ul>
+
-
<ul>
+
-
<li>Observations & Results: <br>We now seemed to have found the mistake that caused problems with the loss of DNA material in the second mutagenesis round! Miscommunication led to using the wrong kit for purification steps. We decided to use the peqGOLD Cycle-pure Kit (PeqLab) instead of the subsequent ethanol precipitation as well, as this seemed to rise fewer problems.</li>
+
</ul>
</ul>
<br></td></tr>
<br></td></tr>
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<td width="900" bordercolor="black" valign="top">
<td width="900" bordercolor="black" valign="top">
<h2><b>V08_17 </b></h2><br>
<h2><b>V08_17 </b></h2><br>
-
<b>V08_17_1 2<sup>nd</sup> round: <i>Dpn</i>I/<i>Bsa</i>I digest and purification</b><br>
+
<b>V08_17_1 2<sup>nd</sup> round: Analysis of the transformation plates and liquid cultures</b><br>
-
<ul>
+
-
<li>Experiment: <br>The PCR purification was performed using the peqGOLD Cycle-pure Kit (PeqLab), modified using columns from the peqGOLD Plasmid Miniprep Kit (PeqLab)! Elution in 100 µL EB.</li>
+
-
</ul>
+
-
<ul>
+
-
<li>Observations & Results: <br>We now seemed to have found the mistake that caused problems with the loss of DNA material in the second mutagenesis round! Miscommunication led to using the wrong kit for purification steps. We decided to use the peqGOLD Cycle-pure Kit (PeqLab) instead of the subsequent ethanol precipitation as well, as this seemed to rise fewer problems.</li>
+
-
</ul>
+
-
<br>
+
-
<b>V08_17_2 2<sup>nd</sup> round: Ligation</b><br>
+
<ul>
<ul>
-
<li>Experiment: <br>The PCR purification was performed using the peqGOLD Cycle-pure Kit (PeqLab), modified using columns from the peqGOLD Plasmid Miniprep Kit (PeqLab)! Elution in 100 µL EB.</li>
+
<li>Experiment: <br>Both the liquid culture and the plates were investigated. The liquid culture was centrifuged at 4 °C, 4800 rpm for 10 min. The cell pellet was stored at -20 °C.</li>
</ul>
</ul>
<ul>
<ul>
-
<li>Observations & Results: <br>We now seemed to have found the mistake that caused problems with the loss of DNA material in the second mutagenesis round! Miscommunication led to using the wrong kit for purification steps. We decided to use the peqGOLD Cycle-pure Kit (PeqLab) instead of the subsequent ethanol precipitation as well, as this seemed to rise fewer problems.</li>
+
<li>Observations & Results: <br>Both the liquid culture and the plates exhibited bacterial growth. Plates were as follows:<br>
 +
10<sup>4</sup> 31 colonies<br>
 +
10<sup>5</sup> 2 colonies<br>
 +
10<sup>6</sup> 0 colonies<br>
 +
</li>
</ul>
</ul>
<br></td></tr>
<br></td></tr>

Latest revision as of 17:54, 26 September 2012

Deutsch  / English 

#3 Chemoreceptor Library - 16th Week

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V08_13


2nd round: Mutagenesis PCR, 1000 µL
  • Experiment:
    The PCR was set up in a volume of 1000 µL following the protocol from week 10.


V08_14


2nd round: PCR purification
  • Experiment:
    The PCR purification was performed using the peqGOLD Cycle-pure Kit (PeqLab), modified using columns from the peqGOLD Plasmid Miniprep Kit (PeqLab)! Elution in 100 µL EB.
  • Observations & Results:
    We now seemed to have found the mistake that caused problems with the loss of DNA material in the second mutagenesis round! Miscommunication led to using the wrong kit for purification steps. We decided to use the peqGOLD Cycle-pure Kit (PeqLab) instead of the subsequent ethanol precipitation as well, as this seemed to rise fewer problems.


V08_15


V08_15_1 2nd round: DpnI/BsaI digest and purification
  • Experiment:
    The digest was performed according to protocol from week 10. The subsequent purification was carried out using the peqGOLD Cycle-pure Kit (PeqLab)!!!
  • Observations & Results:
    The corresponding agarose gel showed band of the expected sizes! At last, there did not seem to be any loss of DNA material.

V08_15_2 2nd round: Ligation, 2000 µL
  • Experiment:
    The ligation was performed according to the establoshed protocol from week 10. Incubation over night at 16 °C.


V08_16


V08_16_1 2nd round: Purification of ligation from V08_15
  • Experiment:
    The purification of the ligation was now done using the peqGOLD Cycle-pure Kit (PeqLab) with columns from peqGOLD Plasmid Miniprep Kit I (PeqLab) instead of an ethanol precipitation. This way we could be sure to gain enough DNA material to go on with our library generation. Elution in 50 µL EB.
  • Observations & Results: The corresponding agarose gel FINALLY showed a band of the expected size, indicating that we could purify our DNA material successfully.

V08_16_2 2nd round: Transformation of electrocompetent cells with mutant plasmid mixture
  • Experiment:
    Electrocompetent cells were prepared according to protocol. Transformation was performed acording to the established protocol from week 10.


V08_17


V08_17_1 2nd round: Analysis of the transformation plates and liquid cultures
  • Experiment:
    Both the liquid culture and the plates were investigated. The liquid culture was centrifuged at 4 °C, 4800 rpm for 10 min. The cell pellet was stored at -20 °C.
  • Observations & Results:
    Both the liquid culture and the plates exhibited bacterial growth. Plates were as follows:
    104 31 colonies
    105 2 colonies
    106 0 colonies


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